Cryptosporidium causes severe diarrhea in infants and chronic diarrhea in immunosuppressed persons. There are no human vaccines against the parasite, and the primary drug used to treat Cryptosporidium is not effective in immunosuppressed persons. The goal of this R21 proposal is to determine whether synthesis of the oocyst wall and/or development of infectious sporozoites are necessary for maintaining chronic infections by Cryptosporidium. Briefly, oocysts contain four haploid sporozoites, which infect epithelial cells and reproduce asexually as merozoites. Merozoites differentiate into sexual forms, which fuse to form diploid zygotes. Zygotes then make the oocyst wall, within which meiosis occurs and four sporozoites develop. Because asexual reproduction by Cryptosporidium is time-limited, autoinfection by oocysts appears to be necessary for maintaining severe or chronic infections. The innovation and risk here is to combine biochemical and morphological studies of the oocyst wall, which is a specialty of our lab, with recently developed methods for genetically manipulating Cryptosporidium in mice and for maintaining indefinitely the entire Cryptosporidium life cycle in hollow fiber cultures of HCT-8 cells. The advantages of genetically manipulating Cryptosporidium in hollow fiber cultures are 1) oocysts are obtained without contamination by stool bacteria and 2) survival surgeries to implant electroporated trophozoites into the mouse intestine are avoided. In addition, we propose to co-infect knockouts with wild-type parasites to 1) make diploid zygotes useful for identification of essential genes for oocyst wall synthesis and/or sporulation and 2) produce sporozoites with these essential genes knocked out. In the first Specific Aim we will knock out genes involved in oocyst wall formation (e.g. xylan synthase that forms fibrils in the inner layer of the wall or polyketide synthase that makes acid-fast lipids) and determine the effects on oocyst wall formation and sporozoite production in hollow fiber cultures. We will also knock out genes involved in meiosis, which is the first step to produce four infectious sporozoites within oocyst walls. The knockouts will tell us what genes are essential for development of oocyst walls and sporozoites and, if autoinfection is blocked, suggest novel drug targets to prevent chronic infection. In the second Specific Aim we will infect sets of mice with knockout sporozoites and wild-type controls and monitor the infections using non-invasive in vitro imaging system (IVIS). If the gene knockouts prevent oocyst wall formation and sporulation, we expect that autoinfection will be blocked, and infections will be time-limited compared to the reporter line. We will also compare infection of human 3D intestinal cell tissues by genetically manipulated oocysts and sporozoites.